A number of viral and non-viral (synthetic) methods for gene transfer have been developed that are intended for gene therapy (Mulligan, Science, 260, 926 to 932 (1993) and Ledley, Human Gene Therapy, vol. 6, 1129 to 1144 (1995)). In general, viral methods are more effective than non-viral methods for gene delivery into cells. However, viral-vector may present safety concerns due to the concurrent introduction of essential gene elements from the parent virus, leaky expression of viral genes, immunogenicity, and modification of the host genome structure. In general, a non-viral vector has less cytotoxicity and less immunogenicity. However, a majority of non-viral methods have a lower gene transfer efficiency, esp. in vivo, than some viral vectors.
Thus, both virus vectors and non-viral vectors have limitations as well as advantages. Therefore, a high-efficiency and low-toxicity gene transfer vector for in vivo use must be developed so as to compensate for the limitations of one type of vector system with the advantages of another type of system.
On the other hand, HVJ has high immunogenicity, and is known to induce CTL especially when NP protein is produced in a large quantity (Cole, G. A. et al., J. Immunology 158, 4301 to 4309 (1997)). It is also feared that the protein synthesis by the host may be inhibited.
HVJ also has a problem in that particles which are created by a method in which a fusion protein is purified by subjecting the virus fusion protein to centrifugation or column manipulation so as to be reconstituted on a lipid membrane may lose the other proteins (primarily M protein) of the virus due to the reconstitution, so that the ratio between the F1 which is required for fusion activity and the HN protein cannot be maintained at the same level as that of the wild-type virus, resulting in a lower fusion activity. Moreover, since the orientation in which the fusion protein is inserted into the lipid membrane at the time of reconstitution may not necessarily be the same as in the wild-type virus, some unknown antigens may be presented.
A method has also been reported in which reconstitution is carried out by adding new molecules (Uchida, T. et al., J. Cell. Biol. 80, 10 to 20, 1979). However, this method runs a high risk of losing the original viral functions because the membrane composition of the completed particles is substantially different from that of the native virus particles.
Methods which involve encapsulating genes or proteins in liposomes and fusing this with inactivated HVJ to create fusion particles, as in conventional HVJ-liposome, have enabled a non-invasive gene transfer into cultured cells or in vivo tissue. This technique is in frequent use worldwide at the animal experimentation level (Dzau, V. J. et al., Proc. Natl. Acad. Sci. U.S.A., 93, 11421 to 11425 (1996) and Kaneda, Y. et al., Molecular Medicine Today, 5, 298 to 303 (1999)). However, it has also been found that this technique has drawbacks: for example, the procedure may be complicated because two different vesicles, i.e., a virus and a liposome must be prepared; the particles whose average diameter has increased to be 1.3 times that of viral particles due to fusion with the liposome have a fusion activity which in 10% or less of that of the virus.
Furthermore, with respect to some tissue, vectors based on conventional HVJ may not be able to achieve any gene transfer, or if at all they do, with an extremely low efficiency. This indicates that the tissue for gene therapy based on conventional methods may be limited.
There is a desire for the development of a viral vector for human gene therapy, which can be prepared safely, highly efficiently, and simply, and yet enables gene transfer to a broad range of in vivo tissue.
Therefore, an objective of the present invention is to develop a safe, highly efficient, and simple virus-based gene transfer vector for a broad range of cultured cells or in vivo tissue which can overcome the drawbacks of conventional reconstituted HVJ vector methods or HVJ-liposome methods.